Method for Producing Natively Folded Proteins in a Prokaryotic Host

Abstract

The present invention relates to a method for producing a protein of interest containing one or more disulfide bonds in its native state. The method comprises that a prokaryotic host cell is genetically engineered to express the protein of interest and a sulfhydryl oxidase in the cytoplasm of the host cell. The protein of interest is formed in a soluble form and contains disulfide bonds due to the presence of the sulfhydryl oxidase in the cytoplasm of said host cell. The present invention relates also to a prokaryotic host cell and a vector system for producing a protein of interest containing natively folded disulfide bonds.

Description

PRIORITY CLAIM[0001]This is a continuation-in-part application of International application number PCT / FI2010 / 05448 filed on Jun. 2, 2010 claiming priority of the Finnish national patent application number 20095615 filed on Jun. 2, 2009, the contents of both of which are incorporated herein by reference in their entirety.SEQUENCE DATA[0002]This application contains sequence data provided in computer readable form and as PDF-format. The PDF-version of the sequence data is identical to the computer readable format.FIELD OF THE INVENTION[0003]This invention relates to a method, a host cell and a vector system for producing a protein of interest containing one or more disulfide bonds in its native state. In particular, the invention relates to a method, a host cell and a vector system for producing such proteins in a prokaryotic host.DESCRIPTION OF RELATED ART[0004]Many proteins and enzymes of biotechnological importance contain structure stabilizing disulfide bonds, with an estimated o...

AI Technical Summary

Benefits of technology

[0076]The advantage of the present invention is that the protein of interest is formed in soluble form. Typically no denaturation and renaturation steps of the protein are needed. Furthermore, the protein is produced directly in biologically active form.

[0077]Commercially significant proteins which may be produced by using the present invention comprise for example insulin, blood coagulation factors, cytokines, chemokines, interferons, growth hormones and single chain antibodies.

[0078]In this disclosure as examples of such proteins are the luminal domain of human tissue factor, E. coli alkaline phosphatase and phytase, bovine pancreatic trypsin inhibitor (BPTI), human colony stimulating factor 3 (CSF3), bone morphogenic protein 4 (BMP4), tissue plasminogen activator (t-PA), interferon α2, interleukin 6, interleukin 17, resistin and growth hormone 1.

[0079]In this disclosure the ability to generate “soluble protein” or “insoluble inclusion bodies” is deduced from SDS-PAGE analysis of total and soluble fractions of a cell lysate. The generation of insoluble inclusion bodies is a common occurrence when proteins that natively contain disulfide bonds are expressed in the cytoplasm of prokaryotic hosts. The formation of disulfide bonds allows protein folding to occur and hence allows the formation of soluble protein.

[0080]In this disclosure the “number of disulfide bonds” is deduced from the total number of cysteines in the protein and the number of cysteines free to react with iodoacetamide, as determined from mass spectrometric analysis after treatment of the protein with iodoacetamide. The reaction of a thiol group with iodoacetamide adds 57 Da to the mass of the protein.

[0081]In this disclosure the “biological activity” of a protein is deduced by well known methods in the art appropriate for the individual proteins being assayed. The biological activity or function of a protein reflects characteristics of the protein that result from the structure and conformational flexibility of the protein. These in turn are often dependent on the formation of native disulfide bonds. Hence biological activity, for example the ability of an enzyme to catalyze a specific enzymatic activity, is a measure of the attainment of the formation of native disulfide bonds within a protein.

Problems solved by technology

Hence, it is unsurprising that native disulfide bond formation is often the rate-limiting step in the folding of proteins in vitro and in vivo.

However, while some of the participants in the cellular process are known, their precise individual roles are still largely confused.

Currently proteins that contain disulfide bonds are difficult for the biotech industry to produce on a large scale.

Due to this the recombinant proteins are unable to attain their native conformation and form insoluble inclusion bodies.

However, it is costly, complex and generally inefficient.

While native E. coli disulfide bond containing proteins fold efficiently in the periplasm, the yields of heterologously expressed proteins are often very low, in part due to the small size of the periplasm.

However, disulfide bond formation is still slow and inefficient.

In addition, these strains are less genetically stable and grow significantly more slowly than wild type strains.

However, there is the corresponding increase in costs associated with the growth of eukaryotic organisms and problems associated with the large scale production of proteins in cell culture.

None of the mentioned patent publications disclose a prokaryotic expression system for the production of natively folded disulfide bond containing proteins.

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